AU2010209265A1

AU2010209265A1 - Method for producing tooth

Info

Publication number: AU2010209265A1
Application number: AU2010209265A
Authority: AU
Inventors: Kazuhisa Nakao; Takashi Tsuji
Original assignee: Organ Technologies Inc
Current assignee: Organ Technologies Inc
Priority date: 2009-01-28
Filing date: 2010-01-14
Publication date: 2011-08-04
Anticipated expiration: 2030-01-14
Also published as: JPWO2010087118A1; US20110306135A1; CA2750072A1; RU2523559C2; WO2010087118A1; CN102300592B; TW201031386A; US8927284B2; SG173037A1; EP2392356A4; CN102300592A; JP5492791B2; RU2011135807A; KR20110123248A; EP2392356A1; AU2010209265B2

Abstract

Disclosed is a method for producing a tooth having a desired length in one direction. Specifically disclosed is a method for producing a tooth having a desired length in one direction, which comprises the steps of: placing a first cell aggregate and a second cell aggregate in the inside of a support while bringing the first and second cell aggregates into close contact with each other; and culturing the first and second cell aggregates in the inside of the support, wherein the first cell aggregate is composed of one of mesenchymal cells or epithelial cells and the second cell aggregate is composed of the other, and the size of the tooth is controlled by adjusting the length of contact between the first cell aggregate and the second cell aggregate in one direction.

Description

[DESCRIPTION] [Title of Invention] Method for Producing Tooth [Technical Field] 5 [0001] The present invention relates to a method for producing a tooth having a desired size. [Background Art] [0002] 10 The toothisanorganhavingenamel as its outermost layer, a hard tissue called dentin inside the layer, and furthermore, odontoblasts forming the dentin on the inner side with dental pulp in the center. Teeth may be lost due to tooth decay and periodontal disease, but because the presence of teeth has a 15 big impact on one's appearance and on the taste of foods, the concern about tooth reproduction techniques has been increasing. Furthermore, concern towards about tooth reproduction techniques has also been increasing for reasons such as maintaining health and maintaining a high quality of life. 20 Atoothisafunctionalunitthatisformedbytheinducement of the generation process during the fetal period, and is formed by a plurality of cell species. A tooth is not generated by a stem cell system wherein cell species are generated from stem cells such as hematopoietic stem cells and mesenchymal stem 25 cells in adults, and currently, teeth therefore cannot be regenerated with only transplantation of stem cells (stem cell transplantation) achieved by regenerative medicine. Although regeneration of teeth through identification of genes found 1 specifically in the generation process of teeth and artificial inducementofatoothgermhasbeenexamined, complete inducement of regeneration of teeth cannot be achieved only by identifying the genes. 5 [0003] Thus, in recent years, a method for obtaining regenerated teeth by reconstructing the tooth germ using an isolated tissue and cell derived from the tooth germ, and then transplanting the reconstructed tooth germ has been examined. 10 [0004] The present inventors figured out that by arranging a first cell mass and a second cell mass in contact with each other inside a support carrier made from collagen gel, wherein at least either the first cell mass is formed substantially 15 from only one of either mesenchymal cells or epithelial cells derived from the tooth germ or the second cell mass is formed substantially from only the other type of cells, and then by culturing the first and the second cell mass inside the support carrier, cell differentiation can be induced effectively, and 20 it is possible to produce a regenerated tooth germ and regenerated tooth having a specific cell arrangement and directionality (for example, see Patent Literature 1). [0005] Furthermore, the present inventors showed that a 25 regenerated tooth germ and regenerated tooth having a specific cell arrangement and directionality can similarly be obtained even by using oral epithelial cells and their first stage cultured cells as epithelial cells (for example, see Patent 2 Literature 2), or by using amnion-derived cells as mesenchymal cells (for example, see Patent Literature 3), or else by using cells obtained by differentiation inducement of totipotent stem cells as mesenchymal cells (for example, see Patent Literature 5 4) . [0006] Also, in a regenerated tooth germ and regenerated tooth, the size of the tooth differs depending on its position and this size also varies with individuals. Therefore, it is 10 important to control the size from the point of view of regenerating a tooth suitable to the position of the lost tooth. However, in the above documents, the control method for the size of regenerated teeth has not been examined. Furthermore, a set of regenerated teeth may be also obtained by the above 15 method. In such a case, each tooth is separated from the set and used as a graft, but the dif ficulty in controlling the number of teeth and also the size of each tooth included in the set of teeth is easily expected from the point of view of insufficient development of the three-dimensional cell operation technology 20 and insufficient understanding of the mechanism of form control in development biology. [0007] The method for inoculating a cell mixture of a tooth germ including mesenchymal cells derived from the tooth pulp that 25 form the dental bulb and dentin, and also including epithelial cells that contribute to the formation of the enamel in a scaffold made by solidification of a biodegradable polymer made from a copolymer of polyglycolic acid and polylactic acid, and then 3 transplanting it into the body of an animal to form a tooth is also proposed as a method for producing regenerated teeth having the desired size and shape. In this method, the control of the shape of the tooth has been tested by using a scaffold 5 ofthe desiredshape. However, the regeneratedtooth is derived from a tooth germ made from an epithelial cell layer and a mesenchymal cell layer, and the tooth germ is known to grow due to the chronological epithelical-mesenchymal interaction that occurs between the epithelial cells and mesenchymal cells. 10 Thus, if a scaffold is used, sufficient cellular interaction is not obtained. Therefore, the use of a scaffold may not be preferable (for example, see Non-Patent Literature 1). Furthermore, the speed of formation of the tooth is faster than the time taken for the scaffold to decompose. Therefore, the 15 toothmaybe formedwith some part of the scaffoldmixed therein, and it is expected that the reproducibility of the cell arrangement and tooth shape may not necessarily be high. [0008] On the other hand, it was believed that in general, the 20 normal shape of the tooth crown cannot be obtained unless the mesenchymal tissue in the reconstructed tooth germ is perfect. However, it has been reported that even if a mesenchymal cell mass (mass obtained by centrifugal processing after separating the tissues with enzyme treatment) is used in place of 25 mesenchymal tissue, when the number of cells is larger, a comparatively larger size of tooth germ is obtained in the in vitro culture, and the number of tooth cusps is also increased (for example, seeNon Patent Literature 1). However, evenafter 4 transplanting this tooth germ inside a living organism, the shape of the tooth crown and the number of tooth cusps change as compared to a normal tooth, and a tooth with the correct shape is not obtained. According to the report, a tooth with 5 a correct shape is not obtained even by reconstructing through the combination of an epithelial cell mass and a mesenchymal cellmass. Accordingtothe report, in the reconstructing using an epithelial tissue and a mesenchymal cell mass, the correct shape can be created if mesenchymal tissue can be used, however, 10 the limitation imposed during the producing of teeth regarding the use of unified mesenchymal cells indicates that partial resolution is achieved by increasing the number of cells. [0009] Furthermore, it has been reported that when a 15 reconstructed tooth germ is produced with an epithelial tissue and a mesenchymal cell mass, the number of produced teeth increases when the number of mesenchymal cells is increased, however, the size of the teeth is not affected (Non-Patent Literature 2). In the report, the final size of a tooth and 20 tooth cusp is determined by intrinsic factors of the mesenchymal tissue and epithelial tissue, that is, it is concluded that the mesenchymal cells and epithelial cells have an intrinsic memory concerning the final size of a tooth and tooth cusp, respectively. 25 [0010] Furthermore, the present inventors figured out a method for using as many epithelial tissues in the region configuring the enamel knot as the desired number of teeth to form an 5 epithelial cell aggregate, when producing a reconstructed tooth germ using an epithelial cell aggregate and a mesenchymal cell aggregate as a method for controlling the number and form of the teeth to be produced (Patent Literature 5) . According to 5 the method, an aggregate of teeth having a desired number of teeth can be obtained. However, as many epithelial tissues as the number of the desired teeth must be acquired to constitute the region configuring the enamel knot. Furthermore, Patent Literature 5 does not examine the control of the size of a tooth. 10 [Related art] [Patent Literature] [0011] [Patent Literature 1] WO 2006/129672 15 [Patent Literature 2] Japanese Published Unexamined Patent Application No. 2008-29756 [Patent Literature 3] Japanese Published Unexamined Patent Application No. 20 2008-206500 [Patent Literature 4] Japanese Published Unexamined Patent Application No. 2008-200033 [Patent Literature 5] 25 Japanese Published Unexamined Patent Application No. 2008-29757 [Non Patent Literature] [0012] 6 [Non Patent Literature 1] Hu et al. Tissue Engineering Volume 12, Number 8, 2006, 2069-2075 [Non Patent Literature 2] 5 J. Cai et al. Developmental Biology 304 (2007) 499-507 [Summary of the Invention] [Problems to be Solved by the Invention] [0013] Teeth have a size and shape suitable to their function 10 depending on the site at which they grow, and the size and shape are different depending on the site even for the same molar tooth. Furthermore, the size of a tooth varies from individual to individual. Therefore, when producing a regenerated tooth germ and a regenerated tooth as a treatment for lost teeth, 15 it is very important to control its size such that it can function appropriately in the individual where the tooth is transplanted. Furthermore, according to the experience of the inventors, the size of the regenerated tooth germ can change by various conditions, and because of the "memory" specific to the cells, 20 teeth with good reproducibility and the same size are not obtained. [0014] Therefore, an objectof thepresent invention is toprovide a method for producing a tooth having a desired size, in 25 particular, a tooth of which the width of a tooth crown is a desired length. [Solving Means] [0015] 7 As a result of intense studies to resolve the above problems, the present inventors found that the width of the crown of a regenerated tooth depends on the contact length in the predetermined direction of the mesenchymal cell aggregate 5 and the epithelial cell aggregate in the support carrier, and does not depend on the number of each of these cells, and therefore, by adjusting this contact length, the width of the crown of the regenerated tooth can be controlled. Furthermore, it was found that when the mesenchymal cell aggregate and 10 epithelial cell aggregate were respectively formed in an almost column shape, then by controlling a contact length of the axial direction of the column, a tooth having a desired length in one direction can be formed; particularly, by setting the contact length within the range between plus and 15 minus 25% (+25%) of the desired length, a regenerated tooth having a desired length can be obtained; and along with the control of the size of the tooth, the number of tooth cusps can also be controlled; and by setting the contact length to below the predetermined numerical value, a single tooth could 20 be obtained, and thereby the present invention was concluded. That is, the present invention relates to: [1] a method for producing a tooth having a desired length in one direction, comprising: a step of arranging a first cell aggregate and a second 25 cellaggregateinclosecontactinsideasupportcarrier, wherein the first cell aggregate and the second cell aggregate are respectively composed of either one of mesenchymal cells and epithelial cells; and 8 a step of culturing the first and second cell aggregates inside the support carrier, wherein the size of the tooth is adjusted by adjusting a contact length of the predetermined one direction of the first 5 cell aggregate and the second cell aggregate; [2] a method for producing a tooth having a desired length in one direction, comprising: a step of producing a plurality of types of structures in which the first cell aggregate and the second cell aggregate 10 are arranged in close contact inside a support carrier by changing the contact length of the predetermined one direction of the first cell aggregate and the second cell aggregate, wherein the first cell aggregate and the second cell aggregate are respectively composed of either one of mesenchymal cells 15 and epithelial cells; a step of culturing each of the plurality of types of structures inside the support carrier; a step of measuring the length of one direction of the tooth produced in the preceding step and determining a 20 correlation between the length and the contact length; a step of calculating, based on the correlation, the contact length of the first cell aggregate and the second cell aggregate which is required for obtaining a tooth having a desired length in one direction; 25 a step of arranging the first cell aggregate and the second cell aggregate in close contact so as to have the contact length calculated in the preceding step, inside a support carrier, wherein the first cell aggregate and the second cell aggregate 9 are respectively composed of either one of mesenchymal cells and epithelial cells; and a step of culturing the first and second cell aggregates inside the support carrier; 5 [3] a method for producing a tooth having a desired length in one direction, comprising: a step of producing a plurality of types of structures in which an almost column-shaped first cell aggregate and second cell aggregate are arranged in close contact inside a support 10 carriersuchthat theaxialdirectionofeachcolumnisparallel, by changing a contact length of the axial direction of the first cell aggregate and the second cell aggregate, wherein the first cell aggregate and the second cell aggregate are respectively composed of either one of mesenchymal cells and epithelial cells; 15 a step of culturing each of the plurality of types of structures inside the support carrier; a step of measuring the length of one direction of the tooth produced in the preceding step and determining a correlation between the length and the contact length; 20 a step of calculating, based on the correlation, the contact length of the first cell aggregate and the second cell aggregate which is required for obtaining a tooth having a desired length in one direction; a step of arranging the almost column-shaped first cell 25 aggregate and second cell aggregate in close contact inside the support carrier such that the contact length of the axial direction is the length calculated in the preceding step and the axial direction of each column is parallel, wherein the 10 first cell aggregate and the second cell aggregate are respectively composed of either one of mesenchymal cells and epithelial cells; and a step of culturing the first and second cell aggregates 5 inside the support carrier; [4] A method for producing a molar tooth having a desired length in a mesiodistal direction and/or a buccolingual direction, comprising: a step of producing a plurality of types of structures 10 in which an almost column-shaped first cell aggregate and second cell aggregate are arranged in close contact inside a support carrier such that the axial direction of each column is parallel, by changing a contact length of the axial direction of the first cell aggregate and the second cell aggregate and/or a contact 15 length of the direction perpendicular to the axis, wherein the first cell aggregate and the second cell aggregate are respectively composed of either one of mesenchymal cells and epithelial cells; a step of culturing each of the plurality of types of 20 structures inside the support carrier; a step of measuring the length of the molar tooth produced in the preceding step in the mesiodistal direction and/or the buccolingual direction, and then determining a correlation between the contact length of the axial direction and the length 25 of the mesiodistal direction of the molar tooth, and/or a correlation between the contact length perpendicular to the axis and the length of the buccolingual direction of the molar tooth; 11 a step of calculating, based on the correlation, the contact length of the axial direction of the first cell aggregate and the second cell aggregate and/or the contact of the direction length perpendicular to an axis which are required for obtaining 5 the molar tooth having a desired length in a mesiodistal direction and/or a buccolingual direction; a step of arranging the almost column-shaped first cell aggregate and second cell aggregate in close contact inside the support carrier such that the contact length of the axial 10 direction and/or the contact length of the direction perpendicular to the axis is the length calculated by the preceding step and the axial direction of each column is parallel, wherein the first cell aggregate and the second cell aggregate are respectively composed of either one of mesenchymal cells 15 and epithelial cells; and a step of culturing the first and second cell aggregates inside the support carrier; [5] a method for producing a tooth having a desired length in one direction, comprising: 20 a step of arranging an almost column-shaped first cell aggregate and second cell aggregate in close contact inside a support carrier such that the axial direction of each column is parallel, whereby a contact length of the axial direction of the first cell aggregate and the second cell aggregate is 25 approximately within the range between plus and minus 25% of the desired length, wherein the first cell aggregate and the second cell aggregate are respectively composed of either one of mesenchymal cells and epithelial cells; and 12 a step of culturing the first and second cell aggregates inside the support carrier; [6] the method according to the above [5], wherein the step of arranging the first and the second cell aggregates inside 5 the support carrier comprises: a step of producing a plurality of structures in which the first and the second cell aggregates are arranged inside the support carrier; a step of measuring a contact length of an axial direction 10 of the first and the second cell aggregates; and a step of selecting a structure of which the measured contact length is approximately within the range between plus and minus 25% of the desired length. [7] a method for producing a single tooth, comprising: 15 a step of arranging a first cell aggregate and a second cellaggregateinclosecontactinsideasupportcarrier, wherein the first cell aggregate and the second cell aggregate are respectively composed of either one of mesenchymal cells and epithelial cells; and 20 a step of culturing the first and second cell aggregates inside the support carrier, wherein a maximum contact length of the first cell aggregate and the second cell aggregate is equal to or less than a predetermined value. 25 [8] the method according to any one of the above [1] to [7], wherein both the cell aggregates are cell masses; [9] the method according to any one of the above [1] to [8], wherein at least one of the mesenchymal cell and the 13 epithelial cell is derived from a tooth germ; [10] a method for recovering a tooth missing part within an oral cavity, comprising: a step of transplanting the tooth obtained by the method 5 according to any one of the above [1] to [9], into the missing part; [11] the method according to the above [10], wherein the tooth obtained by the method according to any one of the above [1] to [9] is transplanted as is into the missing part without 10 dividing the tooth into two or more parts; [12] themethodaccordingtotheabove [10] or [11], wherein the mesenchymal cell and the epithelial cell are derived from an individual having the missing part; [13] the method according to any one of the above [10] 15 to [12], wherein the oral cavity is an oral cavity of a non-mammal; [14] Amethod for designing a method for producing a tooth having a desired length in one direction under a predetermined condition, 20 wherein the designing method includes a method for determining, when the first cell aggregate and the second cell aggregate in close contact inside a support carrier and the first cell aggregate and the second cell aggregate are respectively composed of either one of mesenchymal cells and 25 epithelial cells, a contact length of a predetermined one direction of the both cell aggregates, which is required for producing a tooth having a desired size, and wherein the method for determining the contact length 14 further includes: a step of producing a plurality of types of structures in which the first cell aggregate and the second cell aggregate are arranged in close contact inside a support carrier by 5 changing the contact length of the predetermined one direction of the first cell aggregate and the second cell aggregate, wherein the first cell aggregate and the second cell aggregate are respectively composed of either one of mesenchymal cells and epithelial cells; 10 a step of culturing each of the plurality of types of structures inside the support carrier; a step of measuring the length of one direction of the tooth produced in the preceding step, and then determining a correlation between the contact length and the length of one 15 direction of the tooth; and a step of calculating, based on the correlation, the contact length of the first and second cell aggregates which is required for obtaining the tooth having a desired length in one direction. 20 [15] amethod for designing amethod for producing a single tooth under a predetermined condition, wherein the designing method includes a method for determining, when the first cell aggregate and the second cell aggregate are arranged in close contact inside a support 25 carrier, a maximum contact length of the both cell aggregates and the first cell aggregate and the second cell aggregate are respectively composed of either one of mesenchymal cells and epithelial cells, which is required forproducinga single tooth, 15 and wherein the method for determining the maximum contact length further includes: a step of producing a plurality of types of structures 5 in which the first cell aggregate and the second cell aggregate are arranged in close contact inside a support carrier by changing the maximum contact length of the first cell aggregate and the second cell aggregate, wherein the first cell aggregate and the second cell aggregate are respectively composed of either 10 one of mesenchymal cells and epithelial cells; a step of culturing each of the plurality of types of structures inside the support carrier; a step of measuring the number of teeth produced in the preceding step and determining the maximum contact length of 15 the first cell aggregate and the second cell aggregate which is required for obtaining a single tooth; and [161 themethodaccordingtotheabove [14] or [151, wherein at least one of the mesenchymal cell and the epithelial cell is derived from a tooth germ. 20 [Effects of the Invention] [0016] According to the present invention, when the mesenchymal cell aggregate and the epithelial cell aggregate are arranged in close contact inside the support carrier, then by adjusting 25 a contact length of the predetermined direction of the mesenchymal cells and epithelial cells, the width of the tooth crown in the contact length direction can be controlled in the regenerated tooth germ and regenerated tooth that are produced. 16 Particularly, when the mesenchymal cell aggregate and epithelial cell aggregate is formed in an almost column shape, then by controlling a contact length of the axial direction of the column, a tooth having a desired length in the axial 5 direction can be formed. Furthermore, it is also possible to design the method for producing a tooth including determining the contact length to enable the production of a tooth having the desired size based on the present invention. 10 Furthermore, according to the method for the present invention, regardless of the number of cells included in each cell mass, if the predetermined contact length can be obtained, a tooth having a desired length can be produced, and therefore, the desired size can be achieved effectively with a fewer number 15 of cells. Furthermore, in the present invention, by setting the contact length between each cell mass to below the predetermined value, a single tooth can be obtained instead of an aggregate of a plurality of teeth. Therefore, instead of passing through 20 the step of segregation, the tooth can be used as is in the form of a graft. [Brief Description of Drawings] [0017] [Fig. 1] 25 Fig. 1 shows the phase contrast microscopy of the organ culture on day zero, the second day, fifth day, and seventh day, when a contact length of the epithelial cell aggregate and mesenchymal cell aggregate during the producing of 17 reconstructed tooth germ is set to less than 450pm, between 450pm and 900pm, and between 900 pm and 1500 pm. The arrow heads in the figure show both ends of the tooth crown region that will form the future tooth crown. 5 [Fig. 2] Fig. 2 is a schematic diagram showing the width of the tooth crown region that will form the future tooth crown, which is measured in the form of indexes showing the size of the regenerated tooth germ on the seventh day of the organ culture. 10 [Fig. 3] Fig. 3 is a bar graph showing the relationship between a contact length of the epithelial cell aggregate andmesenchymal cell aggregate during the producing of reconstructed tooth germ, and the size of the regenerated tooth germ on the seventh day 15 of the organ culture. [Fig. 4] Fig. 4 isagraphshowingtherelationshipbetweenacontact length of the epithelial cell aggregate and mesenchymal cell aggregate during the producing of reconstructed tooth germ, 20 and the size of the regenerated tooth germ on the seventh day of the organ culture. [Fig. 5] Fig. 5 is a schematic diagram showing the width of the tooth crown measured in the form of indexes showing the size 25 of a regenerated tooth generated by transplanting a regenerated tooth germ beneath the subrenal capsule. [Fig. 6] Fig. 6 shows a stereomicroscopic image of a regenerated 18 toothonthe21stdayofthe subrenal capsuleassay, whena contact length of the epithelial cell aggregate and mesenchymal cell aggregate during the producing of reconstructed tooth germ is set to less than 450 pm, between 450 pm and 900 pm, and between 5 900 pm and 1500 pm. The arrow heads in the figure show both ends of the tooth crown. [Fig. 7] Fig. 7 is a bar graph showing the relationship between a contact length of the epithelial cell aggregate andmesenchymal 10 cellaggregateduring theproducingofreconstructedtoothgerm, and the width of the tooth crown of the regenerated tooth on the 2 1 st day of the subrenal capsule assay. [Fig. 8] Fig. 8 isagraphshowingtherelationshipbetweenacontact 15 length of the epithelial cell aggregate and mesenchymal cell aggregate during the producing of reconstructed tooth germ, and the width of the tooth crown of the regenerated tooth on the 2 1 St day of the subrenal capsule assay. [Fig. 9] 20 Fig. 9 is a CT image of a regenerated tooth on the 2 1 st day after the subrenal capsule assay, when a contact length of the epithelial cell aggregate and mesenchymal cell aggregate during the producing of reconstructed tooth germ is set to less than 450 pm, between 450 pm and 900 pm, and between 900 pm and 25 1500 pm. (Fig. 101 Fig. 10 is a graph showing the relationship between a contact length of the epithelial cell aggregate and mesenchymal 19 cell aggregate during the producing of reconstructed tooth germ, and the number of cusps of the regenerated tooth after 21 days of the subrenal capsule assay. [Fig. 11] 5 Fig. 11 shows the results of measurement of the width of tooth crown region of a regenerated tooth germ obtained when a contact length of the cylindrical epithelial cell aggregate and mesenchymal cell aggregate is set to a fixed range in the reconstructed tooth germ, and the number of cells included in 10 each aggregate is changed. [Fig. 12] Fig. 12 shows the results of measurement of the width of tooth crown of a regenerated tooth obtained when a contact length of the cylindrical epithelial cell aggregate and 15 mesenchymal cell aggregate is set to a fixed range in the reconstructed tooth germ, and the number of cells included in each aggregate is changed. [Fig. 13] Fig. 13 shows the results of measurement of the number 20 of cusps of a regenerated tooth obtained when a contact length of the cylindrical epithelial cell aggregate and mesenchymal cell aggregate is set to a fixed range in the reconstructed tooth germ, and the number of cells included in each aggregate is changed. 25 [Description of Embodiments] [0018] A method for producing a desired tooth according to the present invention includes: a step of arranging a first cell 20 aggregate and a second cell aggregate composed respectively of either one of mesenchymal cells and epithelial cells in close contact inside a support carrier; and a step of culturing the first and second cell aggregates inside the support carrier, 5 wherein the size of the tooth is adjusted by adjusting a contact length of one direction of the first and second cell aggregates. [0019] In the present invention, a "tooth" refers to a tissue comprising a layer of dentin on the inner side and a layer of 10 enamel on the outer side in continuation, and having a directionality comprising the tooth crown and the dental root. The directionality of a tooth can be identified by the arrangement of the tooth crown and dental root. The tooth crown and dental root can be checked visually based on the shape and 15 histological staining. The tooth crown is a part having a layered structure comprising enamel and dentin, and the dental root does not have the enamel layer. (0020] Dentin and enamel may be easily and morphologically 20 identified by those skilled in the art by histological staining or the like. Furthermore, the enamel can be identified by the presence of enamel blast cells, and the presence of enamel blast cells can be confirmed by the presence of amelogenin. On the other hand, the dentin can be identified by the presence of 25 odontoblasts, and the presence of odontoblasts can be confirmed by the presence of dentin sialoprotein. The confirmation of amelogenin and dentin sialoprotein can be performed easily by methods well known in this field, for example, in situ 21 hybridization and antibody staining can be used. [0021] In the present invention, "tooth germ" and "tooth bud" are expressions used to specifically refer to different stages 5 of generation of teeth. In this case, the tooth germ refers to the initial germ of a tooth that is determined to become a tooth in the future, and indicates from the bud stage to the bell stage that are generally used as the stages of generation of a tooth, and is a tissue in which the accumulation of dentin 10 and enamel, which is characterized as hard tissues of a tooth, are not particularly identified. On the other hand, the "tooth bud" indicates a tissue after the stage of the "tooth germ" used in the present invention, and is a tissue that ranges from the stage when the dentin and enamel accumulation characterized 15 as the hard tissues of the tooth have started to form, up to the stage before the tooth erupts from the gums and generally starts functioning as a tooth. The generation of a tooth from the tooth germ occurs through each of the bud stage, cap stage, and the early bell stage and late bell stage. In the bud stage, 20 theepithelialcellsinfoldintothemesenchymalcellstothicken, and in the cap stage, the epithelial cells are infolded to encompass the mesenchymal cells. When the early bell stage andlatebell stagearereached, theepithelialcellpartbecomes the outer enamel, and the mesenchymal cell part forms the dentin 25 ontheinnerside. Thegenerationofthetoothgermiscontrolled by the cellular interaction between the epithelial cells and mesenchymal cells via cytokine, thereby forming a tooth. [0022] 22 In the present invention, "mesenchymal cells" refer to the cells derived from the mesenchymal tissue as well as the cells obtained by culturing such cells, and "epithelial cells" refer to cells derived from the epithelial tissue as well as 5 the cells obtained by culturing such cells. Furthermore, in the present invention, the "peridontal tissue" refers to the alveolar bone and the periodontal membrane formed mostly on the outer layer of the tooth. Those skilled in the art can easily identify the form of the alveolar bone 10 and periodontal membrane through a histological staining. [0023] In the present invention, the "step of arranging a first cell aggregate and a second cell aggregate composed respectively of either one of mesenchymal cells and epithelial cells in close 15 contact inside a support carrier (hereinafter referred to as the "arrangement step") is, for example, described in Patent Literatures 1 to 5, and its entire disclosure is incorporated herein for the purpose of reference. [0024] 20 The above-mentioned first cell aggregate and the second cell aggregate are formed substantially from only mesenchymal cells or epithelial cells, respectively. "Formed substantially from only mesenchymal cells" implies that in the present invention, one of the aggregates of cells performs the 25 same functions as when composed only of mesenchymal cells, and does not include cells other than mesenchymal cells, to the extent possible. The same applies to "formed substantially from only epithelial cells." 23 Here, cell aggregate refers to cells in close contact, and could even be a cell mass prepared from discrete cells even in the case of tissues. The use of a tissue has the advantage of easily enabling acquisition of teeth with the correct cell 5 arrangement and shape, but the amount that can be obtained is limited. Because cultured cells can also be used as cell mass, they are comparatively easier to obtain and therefore, more preferable. According to the method for the present invention, a regenerated tooth with the correct cell arrangement and shape 10 can be obtained even by using a cell mass. [0025] The mesenchymal cells and epithelial cells configuring the cell aggregate can be derived from any tissue of a living organism as long as a regenerated tooth can be generated from 15 the regenerated tooth germ formed by using these cells. Preferably, from the point of view of effectively forming a tooth having a specific structure and directionality by reproducing the cell arrangement inside the living organism, at least one of these cell aggregates must be derived from the 20 tooth germ. It is more preferable that both the mesenchymal cells and epithelial cells be derived fromthe tooth germ. From the point of view of juvenility and homogeneity of the stage of differentiation of cells, the tooth germ is desired to be in the stage between the bud stage and the cap stage. 25 [0026] Examples of the mesenchymal cells derived from other than a tooth germ include cells derived from other mesenchymal tissues in a living organism. Preferably, these are bone 24 marrow cells and mesenchymal cells not containing blood cells, more preferably, mesenchymal cells in the oral cavity, bone marrow cells inside the jawbone, mesenchymal cells derived from cranial neural crest cells, mesenchymal precursor cells 5 which can differentiate into the mesenchymal cells, and stem cells thereof. As far as mesenchymal cells are concerned, an example of using amnion-derived cells is described in Patent Literature 3, and an example of using cells obtained by differentiation inducement of totipotent stem cells is 10 described in Patent Literature 4 as mesenchymal cells, and its entire disclosure is incorporated herein for the purpose of reference. [0027] The epithelial cells may also be those derived from other 15 than a tooth germ, and examples thereof include cells derived from other epithelial tissues in a living organism. Preferable examples of the epithelial cells include epithelial cells of skin, mucosa and gingiva in the oral cavity, and more preferable examples of the epithelial cells include immature epithelial 20 precursorcellswhichcangenerate differentiated, forexample, keratinized or parakeratinized, epithelial cells such as skin and mucosa. Examples of such immature epithelial precursor cells include non-keratinized epithelial cells and stem cells thereof. An example of using oral epithelial cells and their 25 first stage cultured cells as epithelial cells is described in Patent Literature 2, and its entire disclosure is incorporated herein for the purpose of reference. [0028] 25 A tooth germ and other tissues may be collected from the jawbone or the like of various animals such as dogs and cats besides primates such as humans and monkeys and ungulates such as pigs, cows and horses, which are mammals; and rodents such 5 as mice, rats and rabbits, which are small mammals. For the collection of the tooth germ and the tissue, a condition generally used for collecting a tissue may be applied without modification, and the tooth germ and the tissue may be collected under sterile conditions and stored in an appropriate 10 preservation solution. Examples of a human tooth germ include the tooth germ of a third molar, which is the so-called wisdom tooth, as well as a fetal tooth germ, and, from the point of view of utilization of autogenous tissues, usage of the tooth germ of a wisdom tooth is preferable. In the case of mice, 15 the tooth germ of a mouse with a fetal age of 10 to 16 days may be used. [0029] During the preparation of the mesenchymal cells and epithelial cells from the tooth germ, the tooth germ isolated 20 from its surrounding tissue is first divided into a tooth germ mesenchymal tissue and a tooth germ epithelial tissue based on their shapes. To facilitate isolation, an enzyme may be used at this time. Examples of the enzyme include dispase, collagenase and trypsin. 25 [0030) The cell masses according to the present invention indicate a mass of cells derived from the mesenchymal tissue or epithelial tissue, and may be prepared by aggregating the 26 cells obtained by dispersing the mesenchymal tissue or epithelial tissue, or by aggregating the cells obtained from the first stage or passage culture of the cells. (0031] 5 Enzymes, such as dispase, collagenase and trypsin may be used to disperse the cells. To obtain a sufficient number of cells, a medium generally used for animal cell culture, such as Dulbecco's Modified Eagle Medium (DMEM), may be used as the medium for the culture during the first stage or passage culture 10 of the dispersed cells prior to the preparation of the cell mass. A serum for promotion of cell growth may be added, or, as an alternative to the serum, a cellular growth factor such as FGF, EGFor PDGFora knownserumcomponent suchas transferrin may be added. In cases where serum is added, its concentration 15 may be changed appropriately depending on the culture state, and may usually be about 10%. For the cell culture, normal culture conditions, such as those for culture in an incubator at 370C under 5% C0 2 , may be applied. An antibiotic such as streptomycin may be added as appropriate. 20 [0032] To aggregate cells, centrifugal processing is performed for the cell suspension. When the mesenchymal cell mass and epithelial cell mass are formed in close contact, they must be maintained respectively at a high density to ensure the cell 25 interaction. A high density state means a density almost equivalent to the density at which a tissue is constructed, for example, the high density is in the range of 5x107 cells/ml to 1x109 cells/ml, preferably 1x108 cells/ml to 1xl09 cells/ml, 27 and most preferably 2x108 cells/ml to 8x108 cells/ml. The methods for preparing a cell mass having such a high cell density are not limited, for example, cells may be aggregated and precipitated by centrifugation. The centrifugal processing 5 is particularly desired since this conveniently enables achievement of the high density without impairing the cell activity. Such centrifugation may be carried out at a revolution speed equivalent to a centrifugal force of 300xg to 1200xg, and preferably 500xg to 1000xg, for three minutes 10 to ten minutes. Centrifugation at lower than 300xg may not be able to increase the cell density sufficiently, while centrifugation at higher than 1200xg may cause damage to the cells. [0033] 15 In cases where high density cell masses are prepared by centrifugation, the centrifugation is normally carried out after preparing a suspension of cells in a container such as a tube used for cell centrifugation, and the supernatant is removed to the greatest extent possible, leaving the cells as 20 the precipitates. Here, the volume of components other than the cells of interest (for example, a culture medium or a buffer solution) is preferably not more than the volume of the cells, andmost preferably, components other than the cells of interest are not contained. If such high density cell aggregates are 25 in close contact with each other inside the support carrier according to the method described later, a closely-packed cell is obtained, and the cell interaction is effectively exerted. [0034] 28 The support carrier used in the present invention may be one in which cells maybe cultured, and ispreferablyamixture with the above-described medium. The material of the support carrier is not particularly limited, for example, collagen, 5 agarose gel, carboxymethyl cellulose, gelatin, agar, hydrogel, Cellmatrix (trade name), Mebiol Gel (trade name), Matrigel (trade name), elastin, fibrin, laminin, extracellular matrix mixture, polyglycolic acid (PGA), polylactic acid (PLA), and lactic acid/glycolic acid copolymer (PLGA) may be used. These 10 support carriers may have a hardness with which the cells can be virtually maintained at the locations where the cell aggregates were positioned in the support carrier, and examples of these support carriers include those in the forms of a gel, fiber and solid. Of these, materials having the appropriate 15 hardness and retention, such as collagen, agarose gel, carboxy methyl cellulose, gelatin, agar, hydrogel, Cellmatrix, Mebiol Gel, Matrigel, extracellular matrix mixture, elastin, fibrin, and laminin are preferable. In this case, the hardness with which the cells can be virtually maintained at their locations 20 maybehardnesswhichisapplicable tothree-dimensionalculture, that is, a hardness with which the position of the cells can be maintained while hypertrophy of the cells due to their growth is not inhibited, and such hardness can be easily determined by those skilled in the art. 25 [0035] Furthermore, the support carrier used in the present invention may have a retention whereby the cells can maintain their close contact of cell aggregate without being dispersed. 29 The "close contact" indicates that the above-mentioned high density mesenchymal cell aggregate and epithelial cell aggregate maintain the same level of density even in the vicinity of the contact surface of the mesenchymal cells and epithelial 5 cells. If the support carrier that can retain the close contact is collagen, for example, its usage at the final concentration of 2 mg/ml to 3 mg/ml, that is, at a jelly strength of 120 g to 250 g according to the method conforming to JIS-K6503-1996 (measured as the load required to push down by 4 mm with a 12. 7-mm 10 dia. plunger) provides an appropriate hardness. Even in the case of other types of support carriers, if similar strength is obtained with the similar evaluation method, it can be used as the support carrier in the present invention. Furthermore, by combining together one or more types of support carriers, 15 a support carrier with a hardness equivalent to the desired jelly strength may also be obtained. [00361 The methods of arranging the first cell aggregate and second cell aggregate inside the support carrier are not 20 particularly limited, but if the cell aggregates is a cell mass, for example, the precipitate obtained above by centrifugation may be fed inside the support carrier with a micro syringe, etc., and arranged. If the cell aggregate is a tissue, it can be arranged at any position inside the support carrier by using 25 the tip of the syringe needle. [0037] In the present invention, the methods of arranging the first cell aggregate and the second cell aggregate in the support 30 carrier in close contact with each other is not particularly limited, for example, after arranging one of the cell aggregates in the support carrier, the other cell aggregate may be positioned such that it presses against the first cell aggregate, 5 and thus both can be set in close contact with each other. More specifically, by appropriately changing the position of the tip of the above-mentioned syringe needle in the support carrier, one of the cell aggregates can be made to press against the other cell aggregate. When using an epithelial tissue or a 10 mesenchymal tissue as the cell aggregate, the surface of the tissue that was in contact with the mesenchymal tissue or the epithelial tissue in the original tooth germ may be arranged such that it is in contact with the other cell aggregate. Further, after the arrangement, it is also preferable 15 to set upa step of solidification of the support carrier. This enables the cell to further aggregate thus resulting in a higher-density state. For example, when collagen gel is used, solidification can be achieved by leaving to stand at the culture temperature for several minutes to several tens of minutes. 20 By this, components inside the cell aggregate other than the cells are reduced to the minimum possible extent, and a higher-density state is achieved. [0038] In the present invention, the "step of culturing the first 25 and the second cell aggregate inside the support carrier (hereinafter referred to as the "culturing step") is described in the Patent Literatures 1 to 5, and its entire disclosure is incorporated herein for the purpose of reference. 31 [0039] The culturing period varies depending on the number of cells positioned in the support carrier and the states of the cell masses, as well as on the conditions under which the 5 culturing step is carried out, and the type of animal, and those skilled in the art can appropriately select the time period. In the case of transplantation inside the oral cavity, a minimum of one day culturing period, and more preferably, a period of three days or more is desired to enable a functional tooth to 10 erupt. By increasing the length of the culturing period, a great deal of progress can be made in the formation of a reconstructed tooth germ including the formation of accumulations of dentin and enamel, formation of the tooth crown, and formation of the 15 dental root. To achieve the desired state, for example, culturing can be performed for 6 days or more, 30 days or more, 50 days or more, 100 days or more, or 300 days or more, and the medium and culture conditions can also be changed during culturing. 20 [0040] The culturing step inside the support carrier may be performed only by the support carrier which includes the first and the second cell aggregates, or the culture may be performed in the presence of other animal cells. 25 In cases where the culture is performed onlyby the support carrier, the culture can be performed under normal conditions used for culturing of animal cells. Here, a serum derived from mammals, and various cellular factors which are known to be 32 effective in growth and differentiation of these cells may be added to the culture. Examples of such cellular factors include FGF and BMP. [0041] 5 From the point of view of gas exchange and nutrient supply for the cell aggregates, and also from the point of view of performing the entire steps in vitro without any contact or mixing of other animal cells, it is preferable to use organ culture for culturing inside the support carrier. In organ 10 culture, generally, culturing is performed by floating a porous membrane on a medium suitable for growth of animal cells and placing a support carrier having the first and the second cell aggregates, on the membrane. The porous membrane used herein is preferably a membrane having many pores with a diameter of 15 0.3 to 5 pm, and specific examples thereof include Cell Culture Insert (trade name) and Isopore Filter (trade name). [0042] On the other hand, performing the culture inside the support carrier in the presence of other animal cells enables 20 theearlyformationofatoothhavingaspecificcellarrangement in response to the actions of various cytokines and the like from the animal cells. Such culture in the presence of other animal cells maybe performed by culturing ex vivo using isolated cells or cultured cells, and furthermore, the support carrier 25 having the first and the second cell aggregates may be transplanted into a living organism to carry out culture in vivo. [0043] 33 Such transplantation into and culture in vivo is especially preferable since a tooth and/or a periodontal tissue can be formed at an early stage. Preferable examples of animals which can be used as a living organism include mammals, 5 preferably non-human mammals such as pigs and mice, and the animal is more preferably derived from the same species as that of the tooth germ tissue. In cases where culturing is performed by transplanting into an animal that is not of the same species as the tooth germ tissue, it is preferable to use an animal 10 which was altered to be immunodeficient. In order to develop an organ or tissue of animal cells as normally as possible, examples of a site in a living organism suitable for such in vivo growth preferably include beneath the subrenal capsule, mesentery (omentum), and subcutaneous site. 15 The culturing period after transplantation varies depending on the size of the tooth at the time of the transplantation and the size of the tooth to be developed, and may be typically 3 to 400 days. For example, the time period of transplantation beneath the subrenal capsule is preferably 20 7 to 60 days, although it varies depending on the size of the tooth germ to be transplanted and the size of the tooth to be regenerated. [0044] Ex vivo preculture may be performed prior to the 25 transplantation into the living organism. The preculture strengthens the bonds between cells and the bond between the first and the second cell aggregates to make the cellular interaction stronger. As a result, the cellular interaction 34 can be strengthened, and the total growth period can be shortened. The preculturing period is not particularly limited. For example, a period of three days or more, preferably seven days 5 or more, is preferable since a tooth bud can be developed from a tooth germ during this period and thus the culturing period after the transplantation can be shortened. For example, in the case of transplantation and culture beneath the subrenal capsule, and organ culture as the preculture, the time period 10 of the organ culture is preferably 1 to 7 days. [0045] A tooth generated according to the above-mentioned arrangement step and culturing step has a tooth-specific cell arrangement (structure) having dentin inside and enamel outside, 15 and preferably has directionality, that is, has a tip (tooth crown) and a root of a tooth at the correct position, enabling it to sufficiently function as a tooth. Therefore, the generated tooth can be widely used as an alternative to a tooth. Furthermore, it may be used in research for elucidation of the 20 generation process of a tooth. [0046] Furthermore, by extending the culturing period, in addition to the tooth itself, a periodontal tissue such as the alveolar bone and periodontal membrane, which support and 25 stabilize teeth on the jaw bone can be formed. As a result, the practicality of the tooth after the transplantation can be further improved. Furthermore, only the periodontal tissue can be isolated and used. 35 [0047] The present invention is characterized in that the length of one direction of the tooth thus obtained is adjusted by adjusting a contact length of the predetermined one direction 5 of the first cell aggregate and the second cell aggregate in the above-mentioned arrangement step. The contact length can be adjusted depending on the size, shape, and position of the cell aggregate to be arranged inside the support carrier. For example, when arranging the cell mass 10 insidethe support carrierwithamicro syringe, the size, shape, and position of the cell aggregate can be changed appropriately by changing the diameter of the syringe needle and by moving the tip of the needle inside the support carrier while extruding the cell mass, and a contact length of any optional direction 15 of the two cell aggregates can be adjusted. When using a mesenchymal tissue and epithelial tissue as the cell aggregate, the shape and size of the tissue can be adjusted before arranging it inside the supportcarrier, andbyadjustingtheirarrangement position inside the support carrier, a contact length of the 20 two cell aggregates can be adjusted. [0048] Furthermore, by producing a plurality of types of structures in which the first and the second cell aggregates have been arranged in close contact with each other inside the 25 support carrier, and then measuring a contact length of both cell aggregates and selecting the structure in which the measured contact length is the desired length, a reconstructed tooth germ having the desired contact length can be obtained, and 36 such a step is also included in "adjusting the contact length" in the present invention. The measurement of the contact length can be done, for example, by observing through a phase contrast microscope. 5 [0049] Here, the length of one direction of the tooth refers to the width of the tooth crown in any direction, for example, the width in the buccolingual direction (direction perpendicular to the row of teeth), and the width in the 10 mesiodistal direction (direction parallel to the row of teeth) are ideally adopted, but not limited thereto. The measurement of the width of the tooth crown can be done properly by those skilled in the art. [0050] 15 It is noted that when a regenerated tooth germ is formed by adjusting a contact length of the predetermined one direction of the first cell aggregate and the second cell aggregate, normally, the length of the same direction as the contact length is adjusted in the tooth crown of the generated tooth. 20 [0051] One of the aspects according to the present invention of the method for producing a tooth having a desired length in one direction includes: a step of producing a plurality of types of structures in which the first cell aggregate and the 25 second cell aggregate are arranged in close contact inside a support carrier by changing a contact length of a predetermined one direction of the first cell aggregate and the second cell aggregate; a step of culturing each of the plurality of types 37 of structures inside the support carrier; a step of measuring the length of one direction of the tooth produced in the preceding step so as to determine a correlation between the contact length and the length of one direction of the tooth; and a step of 5 calculating, based on the correlation, a contact length of the first cell aggregate and the second cell aggregate required for obtaining a tooth having a desired length in one direction. [0052] The step of producing a plurality of types of structures 10 in which the first cell aggregate and the second cell aggregate are arranged in close contact inside a support carrier by changing a contact length of one direction of the first cell aggregate and the second cell aggregate, and then the step of culturing each of the plurality of types of structures inside 15 the support carrier can be executed according to the above-mentioned explanation of the arrangement step, the culturing step, and the method for adjusting the contact length. [0053] The correlation between the contact length and the length 20 of one direction of the tooth can be found according to a well-known method or a method conforming to the same. For example, various graphs expressing the relationship between the contact length and the length of the tooth (width of the tooth crown) may be created, or a formula expressing the 25 relationship between the contact length and the length of the tooth may be created. Furthermore, the distribution of the contact length providing the length of one tooth may be examined, and the range of contact length providing the size of a 38 predetermined tooth may be determined. [0054] The step of calculating a contact length of the first cell aggregate and the second cell aggregate required for 5 obtaining a tooth having a desired length in one direction based on the acquired correlation can be executed by inserting the acquired size of the tooth in the above-mentioned formula and graph. [0055] 10 In this way, after determining the required contact length, a tooth with the desired size can be obtained by arranging the first cell aggregate and the second cell aggregate at the desired contact length in close contact with each other inside the support carrier under almost the same conditions as the 15 above-mentioned arrangement step of the plurality of types of structures, and then performing a culture under almost the same conditions as the above-mentioned culturing conditions of the plurality of types of structures. Here, "almost the same conditions" refers to the conditions under which a tooth having 20 the same length in one direction can be obtained with good reproducibility when the contact length is set to the same. In the arrangement step and culturing step for determining the contact length, and in the arrangement step and culturing step for producing a tooth having a desired length in one direction, 25 it is desired, for example, that the culturing conditions, such as the type of the support carrier, temperature, constitution of the medium, and location of the culture (whether an organ culture or an in vivo culture) be the same. 39 Furthermore, when arranging the first and the second cell aggregates inside the support carrier, it is desired that a contact length of the part expected to form the position that must have the predetermined length in the tooth to be generated 5 in the future be the length calculated above. Those skilled in the art can appropriately determine which directions of the contact surface of the first and second cell aggregates will become which directions in the tooth to be generated in the future. For example, when producing a molar tooth in which 10 the length A of the mesiodistal direction is longer than the length B of the buccolingual direction, the contact surface of the first and second cell aggregates must be generally rectangular, and the longer side must form the contact length that provides length A in the mesiodistal direction. 15 [0056] Another aspect of the method for producing a tooth having a desired length in one direction according to the present invention includes: a step of producing a plurality of types of structures in which the almost column-shaped first cell 20 aggregate and the second cell aggregate composed respectively of either one of mesenchymal cells and epithelial cells are arranged in close contact inside a support carrier such that the axial direction of each column is parallel by changing a contact length of the axial direction of the first cell aggregate 25 and the second cell aggregate; a step of culturing each of the plurality of types of structures inside the support carrier; a step of measuring the length of the one direction of the tooth produced in the preceding step so as to determine a correlation 40 between the contact length and the length; and a step of calculating, based on this correlation, a contact length of the first cell aggregate and the second cell aggregate required for obtaining a tooth having a desired length in one direction. 5 (0057] The step of producing a plurality of types of structures with different contact lengths and then performing the culturing step can be executed according to the above-mentioned explanation of the arrangement step, culturing step, and the 10 method for adjusting the contact length. As described above, the correlation between the contact length and the length of one direction of the tooth can be expressed with a formula or graph, and the range of the contact length providing the predetermined tooth length can also be determined. Following 15 this, a contact length of the first and second cell aggregates required for obtaining a tooth having a desired length in one direction can be determined based on these correlations. [0058] In the present invention, the "almost column shape" refers 20 to an elongated shape extending in one direction, such as an almost cylindrical shape and an almost prismatic shape. If the cell aggregate is a tissue, the tissue may be formed in an almost column shape and then arranged inside the support carrier. Furthermore, if the cell aggregate is cell mass, for 25 example, thetipoftheneedleofamicrosyringecanbepositioned inside the support carrier, and the cell mass can be arranged in an almost cylindrical shape inside the support carrier by extruding the cells while moving the tip of the needle. 41 [0059] In this way, after determining the required contact length, a tooth having a desired length in one direction can be obtained by arranging the almost column-shaped first cell aggregate and 5 the second cell aggregate at the contact length in close contact with each other inside the support carrier under almost the same conditions as the above-mentioned arrangement step of the plurality of types of structures, and then performing a culture under almost the same conditions as the above-mentioned 10 culturing conditions of the plurality of types of structures. [0060] Another aspect of the method for producing a tooth according to the present invention is a method for producing a molar tooth having a desired length in a mesiodistal direction 15 and/or a buccolingual direction, and includes: a step of producing a plurality of types of structures in which the almost column-shaped first cell aggregate andthe second cell aggregate composed respectively of either one of mesenchymal cells and epithelial cells are arranged in close contact inside a support 20 carrier such that the axial direction of each column is parallel by changing a contact length of the axial direction and/or a contact length of the direction perpendicular to the axis of the first cell aggregate and the second cell aggregate; a step of culturing each of the plurality of types of structures inside 25 the support carrier; and a step of measuring the length of the molar tooth produced in the preceding step in the mesiodistal direction and/or the buccolingual direction so as to determine a correlation between a contact length of the axial direction 42 and the length of the mesiodistal direction of the molar tooth, and/or a correlation between a contact length perpendicular to the axis and the length of the buccolingual direction of the molar tooth. 5 [0061] As described above, generally, because the width of the buccolingual direction of a molar tooth is longer than the width of the mesiodistal direction, when the cell aggregate is formed inacolumnshape, thewidthofthe toothcrowninthemesiodistal 10 direction can be controlled by controlling the contact length in the axial direction, and the width of the tooth crown in the buccolingual direction can be controlled by controlling the contact length perpendicular to the axis. [0062] 15 A contact length of the axial direction and a contact length perpendicular to the axis may be changed with any method, for example, the length of the column-shaped cell aggregate, the diameter, and the distance between the axes of both cell aggregates may be changed. If the cell aggregate is a tissue, 20 then by forming it into the desired diameter and length before arranging inside the support carrier, and then adjusting its position inside the support carrier, the contact length in the axial direction and the contact length perpendicular to the axis can be changed. Furthermore, if the cell aggregate is 25 a cell mass, for example, when positioning it inside the support carrier with the help of a micro syringe, the diameter of the cell aggregate can be changed by changing the diameter of the needle, and by changing the distance in which the tip of the 43 needle is moved inside the support carrier, the length of the axial direction of the cell aggregate can be changed. Furthermore, after arranging one cell aggregate, by adjusting the position in which another cell aggregate is arranged, the 5 distance between the axes of both cell aggregates canbe changed. By reducing the distance between both axes such that they are pressing against each other, the contact surface of the cell aggregate becomes generally larger, and in this way, a contact length of the axial direction and a contact length of the 10 direction perpendicular to the axis can be changed. [0063] Besides these, the arrangement step, culturing step, and the steps of determining the correlation between the various lengths can be performed according to the earlier-mentioned 15 methods. [0064] In this way, after determining a contact length of the axial direction and/or a contact length of the direction perpendicular to the axis, a molar tooth having a desired length 20 in a mesiodistal direction and/or a buccolingual direction can be obtained by arranging the almost column-shaped first cell aggregate and the second cell aggregate at the contact length in close contact with each other inside the support carrier under almost the same conditions as the above-mentioned 25 arrangement step of the plurality of types of structures, and then performing a culture under almost the same conditions as the above-mentioned culturing conditions of the plurality of types of structures. 44 [0065] Furthermore, another aspect of the method for producing a tooth having a desired length in one direction according to the present invention includes a step of arranging the first 5 and the second cell aggregates in close contact in an almost column shape in the arrangement step such that the axial direction of each column is parallel, and a contact length of the axial direction of the first and second cell aggregates is within the range between plus and minus 25%, preferably ±10% 10 of the above-mentioned desired length. [0066] As described later, the present inventors found that when an almost cylindrical shaped mesenchymal cell mass and epithelial cell mass are arranged in close contact inside a 15 support carrier such that the axial direction of the circular column is parallel, the length of the tooth thus produced depends on a contact length of the axial direction of the circular column. Furthermore, it was found that by setting the contact length to approximately within the range-between plus and minus 25%, 20 preferably to approximately ±10% of the desired length, a tooth in which the width of the tooth crown of the mesiodistal direction is of the desired length could be obtained. Therefore, for example, if a tooth in which the width of the tooth crown of the mesiodistal direction is approximately X pm is to be produced, 25 the first cell aggregate and the second cell aggregate may be formed in an almost column shape, and a contact length of the axial direction may be set between 0.75X pm and 1.25X pm, preferably between 0.9X pm and 1.1X pm. 45 [0067] The method for controlling a contact length of the almost column-shaped first and second cell aggregates can be performed according to the already explained method. 5 Furthermore, instead of preparing a cell aggregate having the desired length in the axial direction, a plurality of types of structures in which the almost column-shaped first cell aggregate and the second cell aggregate are arranged in close contact inside a support carrier may be produced, a contact 10 length of the axial direction of both cell aggregates may be measured, the structure in which the measured contact length is the desired length may be selected, and the structure may be passed through the culturing step. The measurement of the contact length can be done, for example, by observing through 15 a phase contrast microscope. [0068] A method for producing a single tooth according to the present invention includes: a step of arranging a first cell aggregate and a second cell aggregate composed respectively 20 of either one of mesenchymal cells and epithelial cells in close contact inside a support carrier; and a step of culturing the first and second cell aggregates inside the support carrier, wherein the maximum contact length of the first cell aggregate and the second cell aggregate is equal to or less than the 25 predetermined value. [0069] When an unexpected tooth aggregate was obtained according to the method for Patent Literature 1, the present inventors 46 figured out that by controlling a contact length of the first and second cell aggregates, and by controlling the size of a tooth, a single tooth with good reproducibility can be obtained. This is probably because the first enamel knot that stipulates 5 the number of teeth formed from the tooth germ is not formed in a number more than one within the predetermined distance. If a single tooth can be produced, there is no need to perform isolation before transplanting the acquired tooth. [0070] 10 It is noted that in the method for producing a single tooth according to the present invention, the "maximum contact length" refers to the length of the longest straight line from among the straight lines included in the contact surface of the first cell aggregate and the second cell aggregate. 15 [00711 Furthermore, if the method for producing a single tooth according to the present invention is applied to a mouse, a contact length of the first and second cell aggregates is preferably equal to or less than 3000 pm, and more preferably 20 equal to or less than 1500 pm. It is noted that the contact length is preferably equal to or more than 100 pm, and more preferably equal to or more than 200 pm. The contact length can be controlled according to the already mentioned description. 25 [0072] In the present invention, a single tooth refers to the structure of tooth that can be transplanted into a living organism, which is characterized by the presence of a tooth 47 crown, dental root, dentalpulp, anddentinformedin continuity, with a periodontal bone and an alveolar bone formed around each tooth. Those skilled in the art can easily find the number of produced teeth. 5 [0073] A method for recovering a tooth missing part within an oral cavity according to the present invention includes a step of transplanting a tooth produced by the method for producing the tooth, according to the present invention into the tooth 10 missing part. According to this method, a tooth matching the size of the missing part can be produced and transplanted. [0074] In the method for recovering a tooth missing part within anoral cavityaccordingtothepresent invention, it ispossible 15 totransplant a toothgermora tooth inanystage that isproduced in the producing method according to the present invention. If the formation of the tooth crown can be seen, it is preferable to place the tooth crown on the inner side of the oral cavity. If the formation of the tooth crown cannot be seen, it is 20 preferable to arrange the epithelial cell layer of the corresponding part of the tooth crown or the epithelial cell layer of the reconstructed tooth germ towards the inner side of the oral cavity. Furthermore, it is preferable to arrange the open part of the epithelial-mesenchymal cell layer of the 25 reconstructed tooth germ on the opposite side of the inner side of the oral cavity. In this way, the tip of the tooth (the tooth crown) is towards the inner side of the oral cavity and has the same directionality as the surrounding teeth. 48 [0075] The missing part implies a part arranged in the gums due to the loss of teeth, and its shape is not particularly limited. As long as the regenerated tooth germ or tooth can be embedded, 5 there are no particular limitations concerning the missing part and the type of the desired tooth. The missing part is usually located at the jaw bone or the alveolar bone inside the oral cavity. Furthermore, along with the tooth loss, if the alveolar bone mass has also 10 deteriorated, a well-known clinical method for regeneration of the bone to facilitate embedding of the implant, such as the GTR method (Guided Tissue Regeneration) may be used for the missing part to increase the bone mass. After positioning the tooth germ or the tooth in the cavity, it is preferable 15 to stitch the site according to the normal process. [0076] In the method for recovering a tooth missing part within the oral cavity according to the present invention, the animal on which the transplant is to be performed must preferably be 20 of the same species as that from which the tooth germ used for producing of tooth is extracted, and more preferably must be the same individual as that from which the tooth germ is extracted. Mammals, such as human beings, cows, horses, pigs, dogs, cats, and mice can be used as the animal. Non-mammals may also be 25 used. [0077] Furthermore, the present invention also provides a method for designing a method for producing a tooth having a desired 49 length in one direction under a predetermined condition. "Predetermined conditions" imply conditions where the support carrier, medium, and the culturing method have been identified. By executing under the predetermined condition, the producing 5 method designed according to the method for designing a method for producing a tooth having a desired length in one direction under a predetermined condition, a tooth having a desired length in one direction can be obtained. The above-mentioned designing method according to the 10 present invention includes a method for determining, when the first cell aggregate and the second cell aggregate composed respectively of either one of mesenchymal cells and epithelial cells are arranged in close contact inside a support carrier, a contact length of both cell aggregates, which is required 15 for producing a tooth having the predetermined length in one direction. Here, the method for determining the contact length includes: a step of producing a plurality of types of structures in which the first cell aggregate and the second cell aggregate 20 composed respectively of either one of mesenchymal cells and epithelial cells are arranged in close contact inside a support carrier by changing a contact length of the predetermined one direction of the first cell aggregate and the second cell aggregate; a step of culturing each of the plurality of types 25 of structures inside the support carrier; a step of measuring the length of one direction of the tooth produced in the preceding step so as to determine a correlation between the contact length and the size of the tooth; and a step of calculating, based 50 on the correlation, a contact length of the first cell aggregate and the second cell aggregate required for obtaining a tooth having a desired length in one direction. [0078] 5 Furthermore, the present invention also provides a method for designing the method for producing a single tooth under predetermined conditions. The above-mentioned designing method according to the present invention includes a method for determining, when the 10 first cell aggregate and the second cell aggregate composed respectively of either one of mesenchymal cells and epithelial cells are arranged in close contact inside a support carrier, the maximum contact length of both cell aggregates, which is required for producing a single tooth. 15 Furthermore, the method for determining the maximum contact length includes: a step of producing a plurality of types of structures in which the first cell aggregate and the second cell aggregate composed respectively of either one of mesenchymal cells and epithelial cells are arranged in close 20 contact inside a support carrier by changing the maximum contact length of the first cell aggregate and the second cell aggregate; a step of culturing each of the plurality of types of structures inside the support carrier; and a step of measuring the number of teeth produced in the preceding step so as to determine the 25 maximumcontact length of the first cell aggregate and the second cell aggregate required for obtaining a single tooth. [0079] It is noted that the terms used herein are used to explain 51 a specific embodiment, and are not intended to limit the invention. Moreover, the term "include" used herein is intended to mean the presence of a matter described (member, step, element, 5 numeral, etc.) except for acase where adifferent understanding should be exercised in light of context, and does not exclude the presence of a matter other than the above-described matter (member, step, element, numeral, etc.). Unless there is a different definition, the terms used 10 herein (including technical terms and scientific terms) carry the same meaning as that widely understood by those skilled in the art to which the present invention belongs. The terms used herein should be interpreted to carry the meaning integral to that in the present specification and the related technical 15 field, unless a different definition is explicitly provided, and thus, these should not be idealized nor interpreted in an excessive perfunctory meaning. There is a case where an embodiment of the present invention is explained with reference to a schematic drawing, and when 20 the schematic diagram is employed, an exaggerated explanation may be introduced for the purpose of an explicit explanation. Terms of "first", "second", etc., are used to express various elements, andit is understoodthat these elements should not be limited by the terms. These terms are used merely to 25 distinguish between one element and another element. For example, it is possible to describe a first element as a second element, and similarly, the second element as the first element, without departing from the scope of the present invention. 52 [0080] Even in the above-mentioned designing method, the mesenchymal cells and epithelial cells can be derived from any tissue of a living organism as long as a regenerated tooth can 5 be prepared from the reconstructed tooth germ formed by using these cells. Preferably, at least one of these cells must be derived from the tooth germ, and more preferably, both these cells must be derived from the tooth germ. [0081] 10 Hereinafter, the present invention will be explained in detail with reference to examples. However, the present invention can be embodied in various modes, and the present invention should not be interpreted as being limited to the examples described herein. 15 [Examples] [0082] (1) Preparation of Tooth Germ Epithelial Cells and Tooth Germ Mesenchymal Cells The tooth germ was reconstructed to forma tooth. Amouse 20 is used as the experimental model. From an embryo (at the fetal age of 14.5 days) of C57BL/6N mouse (purchased from Japan SLC, Inc.), a mandibular molar tooth germ tissue was removed under the microscope by a conventional method. The mandibular molar tooth germ tissue was washed with 25 Ca 2+, Mg2+ -free phosphate buffer (PBS(-)), and treated at room temperature for two minutes with the enzyme solution which is PBS(-) supplemented with 50 U/ml (final concentration) Dispase (manufactured by BD, Massachusetts, USA). This was followed 53 by washing with DMEM (manufactured by Sigma, St. Louis, MO) supplemented with 10% FBS (manufactured by Invitrogen, Carlsbad, CA) three times. Subsequently, a DNase I solution (manufactured by Takara, Shiga, Japan) was added to a final 5 concentration of 70 U/ml to disperse the tooth germ tissue, and the tooth germ epithelial tissue and tooth germ mesenchymal tissue was surgically separated using a 25 G injection needle (manufactured by Terumo, Tokyo, Japan). The tooth germ epithelial tissue was washed with PBS(-) 10 three times, and treated at 37'C for 30 minutes with the enzyme solution which is PBS(-) dissolved with 100 U/ml (final concentration) Collagenase I (manufactured by Worthington, Lakewood, NJ), and this process was repeated twice. The cells whose precipitate was recovered through centrifugation were 15 treated at 37 0 C for ten minutes with the enzyme solution which is PBS(-) dissolved with 0.25% Trypsin (final concentration) (manufactured by Signma). This was followed by washing cells with DMEM (manufactured by Sigma) supplemented with 10% FBS (manufactured by Invitrogen) three times. Subsequently, a 20 DNase I solution (manufactured by Takara) was added to a final concentration of 70 U/ml to the cells, and a suspension of the tooth germ epithelial cells separated with pipeting was obtained. On the other hand, the tooth germ mesenchymal tissue was 25 washedwithPBS(-) three times, andtreatedat37 Cfor20minutes with the enzyme solution which is PBS (-) dissolved with 100 U/ml (final concentration) Collagenase I (manufactured by Worthington). This was further treated for ten minutes with 54 PBS(-) supplementedwith0.25% Trypsin (manufacturedby Signma) and 100 U/ml Collagenase I (manufactured by Worthington). Subsequently, 70 U/ml DNase I solution (manufactured by Takara) was added to obtain a suspension of the tooth germ mesenchymal 5 cells separated with pipeting. [0083] (2) Preparation of Reconstructed Tooth Germ Next, a reconstruction of tooth germ was carried out using the above-prepared tooth germ epithelial cells and tooth germ 10 mesenchymal cells. In a 1.5 ml micro tube (manufactured by Eppendorf, Hamburg, Germany) to which silicone grease was applied, tooth germ epithelial cells or tooth germ mesenchymal cells suspended in DMEM (manufactured by Sigma) supplemented with 10% FBS (manufactured by Invitrogen) were added, and the 15 cells were collected by centrifugation (for three minutes at 600xg) as precipitates. The supernatant of the culture medium after the centrifugation was removed as much as possible, and centrifugation was carried out again for threeminutes at 600xg, followed by complete removal of the culture medium remaining 20 around the precipitates of the cells under a stereoscopic microscope using GELoader Tip 0.5-20 pl (manufactured by Eppendorf). To a petri dish to which silicone grease was applied, 30 pl of Cellmatrix type I-A (manufactured by Nitta gelatin, 25 Osaka, Japan) was added dropwise to prepare a collagen gel droplet as a support carrier. Tothis solution, theprecipitate obtained after centrifugation of the tooth germ mesenchymal cellswas placedquantitativelyusinga Hamilton syringe (7105KH 55 PT-3, manufactured by HAMILTON, Reno, NV) to prepare a cell mass in the form of cylindrical shaped cell aggregate. Subsequently, tooth germ epithelial cells in the equal amount to the tooth germ mesenchymal cells were arranged in the same 5 way to form a cell mass such that the respective sides of both cell masses are in contact with each other and the axial direction is parallel to the cylindrical shaped cell mass of the tooth germ mesenchymal cells prepared earlier, and a reconstructed tooth germ was prepared. 10 After this, the collagen gel drop was solidified by allowing it to stand for 20 minutes at 37*C, and the bond between the two cell masses was made strong. A culture vessel was prepared such that DMEM (manufactured by Sigma) supplemented with 10% FBS (manufactured by Invitrogen) is in contact with 15 Cell Culture Inserts (PET membrane having a pore size of 0.4 pm; manufactured by BD) . The solidified reconstructed tooth germ was transferred onto the membrane of Cell Culture Inserts in the culture vessel, to carry out organ culture on the Cell Culture Insert with the conventional method at 37*C, 95% RH, 20 and 5% C0 2 . [0084] (3) Analyzing the Size of the Regenerated Tooth Germ with Organ Culture The cylindrical-shaped cell aggregates of the epithelial 25 cells and mesenchymal cells were arranged in contact with each other at a length less than 450 pm, between 450 pm and 900 pm, and between 900 pm and 1500 pm to form three groups of reconstructed tooth germs, and a reconstructed tooth germ was 56 formed through organ culture (Fig. 1) . The contact length between both the cell masses was measured with a phase contrast microscope. To analyze the width of the tooth crown region of the 5 regenerated tooth germ, the width of the tooth crown region that would be the future tooth crown marked with the arrow heads in Fig. 2 was measured on the seventh day of organ culture in the regenerated tooth germ using a phase contrast microscope. The measurement position is also shown with arrows in the 10 photograph on the seventh day in Fig. 1. Themeasurement results are shown in Fig. 3. Fora contact length less than 450 pm, the width of the tooth crown region was 366±103.lpm, for a contact length between 450 pm and 900 pm, the width of the tooth crown region was 584.0±103.3 pm, 15 and for a contact length between 900 pm and 1500 pm, the width of the tooth crown region was 934.9±239.8 pm. This indicates that the longer the contact length of the epithelial cell aggregate and mesenchymal cell aggregate during the formation of the reconstructedtoothgerm, thewider the toothcrownregion 20 on the regenerated tooth germ. Furthermore, Fig. 4 is a scatter chart of the measured values of the contact length and the width of the crown region, and linear approximation is performed for the straight line in the figure with the least square method. The formula 25 expressing the straight line was y = 0.7114x + 133.95. [0085] (4) Analyzing the Size of the Regenerated Tooth by Subrenal Capsule Assay 57 From an eight-week old C57BL/6 mouse under anesthesia, the hair on the back located on top of the kidneys was shaved, the skin and the peritoneum were cut open to about 1 cm, and the kidneys were removed using ring tweezers (manufactured by 5 Natsume, Tokyo, Japan) . The subrenal capsule was cut open by 2 to 3 mm using a blade (manufactured by Feather, Tokyo, Japan) . In the space between the kidneys and the subrenal capsule, the three groups of reconstructed tooth germs with different contact lengths as shown in example (2) were inserted with collagen 10 gel, the kidneys were placed back, and the muscular coat and skin were stitched. The regenerated tooth was extracted on the 2 1 St day after subrenal capsule assay. The regenerated tooth that was extracted is shown in Fig. 6. The part marked with arrow heads 15 in Fig. 6 was measured as the width of the tooth crown using a stereoscopic microscope. The measurement results are shown in Table 1. [Table 1] 58 A I B C Actually Difference in 'Contact length measured value Difference tooth crown in reconstructed of crown width , percentage; size; tooth germ (pm) of regenerated zB-A|/Ax100(%) (B-A) (pm) tooth (pm) 437.91 482.29 44.38 10.13 410.77 529.76 118. 99 28.97 318.38 317.34 !(1.04) 0.33 336.77 386.35 49.58 14.72 460. 00 491. 92 :31.92 6. 94 619.87 762. 57 142.70 23.02 549. 87 578. 28 28.41 5. 17 295.25 3 87. 8 8 92.63 31. 37 446.70 459. 61 12. 91 2. 89 458.72 490. 37 !31. 65 16. 90 511. 48 402. 97 (108.51) 21.22 384.75 502. 64 117. 89 30.64 426.23 468.40 42.17 9.89 924.66 :1017.47 192.81 :10.04 1020.24 1240. 64 220. 40 21.60 996.84 979. 63 (1721) 1. 73 1000.00 1083 81 83.81 8.38 1223.27 1293 17 69. 90 5. 71 915.86 9. 76 73 90 8. 07 The average value of the percentage expressed by D in the table was 13.03, and the standard deviation was 10.00. Furthermore, the width of the tooth crown obtained by 5 dividing the above-mentioned measurement results into three based on a contact length of less than 450 pm, between 450 pm and 900 pm, and between 900 pm and 1500 pm is shown in Fig. 7. For a contact length less than 450 pm, the width of the tooth crown was 497±118.Opm, for a contact length between 450 10 pm and 900 pm, the width of the tooth crown was 727.0±271.4 pm, and for a contact length between 900 pm and 1500 pm, the width of the tooth crown was 1073.9±186.0 pm. This indicates that the longer a contact length of the epithelial cell aggregate and mesenchymal cell aggregate during the formation of the 59 reconstructed tooth germ, the wider the tooth crown on the regenerated tooth. Furthermore, Fig. 8 is a scatter chart of the measured values of the contact length and the width of the tooth crown, 5 and linear approximation is performed for the straight line in the figure with the least square method. The formula expressing the straight line was y = 0.7257x + 272.15. (0086] (5) Analyzing the Number of Cusps of the Regenerated Tooth 10 with a Micro CT Using a 3D micro X-ray CT for experimental animals (manufactured by RIGAKU, Tokyo, Japan), the regenerated tooth generated with the method shown in (4) was photographed at a voltageof 90.0 kv, electric current of 150.0Awith 10 pm/Pixel. 15 The results are shown in Fig. 9. Next, the image was analyzed using i-View (manufactured by RIGAKU, Tokyo, Japan), a 3D image of the regenerated tooth was taken, and the number of cusps of the regenerated tooth wascounted. Ifacontactlengthofthecellmassesofepithelial 20 cells and mesenchymal cells during the producing of the reconstructed tooth germ, and the number of cusps of the regenerated tooth generated beneath the subrenal capsule are plotted and the correlation coefficient is calculated, a strong correlation is seen to exist between the contact length during 25 reconstruction and the number of cusps of the regenerated tooth (R2=0.658) (Fig. 10) . This indicates that the longer a contact length of the epithelial cells and mesenchymal cells during reconstruction, the more the number of cusps in the regenerated 60 tooth. [0087] (6) Analyzing the Reconstructed Tooth Germ by Changing the Number of Cells with Contact Length of the Cell Aggregate 5 within a Fixed Range A contact length of the cell masses was set in the range of300to500pm. Bypreparingacellmassusingacellsuspension of approximately 0. 05 pl capacity with a Hamilton syringe having an internal diameter of 0.330 mm (7105KH PT-3, manufactured 10 by HAMILTON, Reno, NV) as used in example (2), and by preparing a cell mass using a cell suspension of approximately 0.02 pl capacity with a Hamilton syringe having an internal diameter of 0.203 mm (7002KH PT-3, manufactured by HAMILTON), a reconstructed tooth germ in which the number of cells used for 15 the cell masses was changed was prepared. The forms of the regenerated tooth germ and the regenerated tooth formed from this reconstructed tooth germ were analyzed by the methods shown in examples (3), (4), and (5). The width of the tooth crown region of the regenerated 20 tooth germ is shown in Fig. 11, the width of the crown of the regenerated tooth is shown in Fig. 12, and the number of cusps in the regenerated tooth is shown in Fig. 13. Even if the number of cells is changed by keeping the contact length within a fixed range, no significant change was observed in the forms of the 25 regenerated tooth germ and the regenerated tooth. 61